Recombinant pichia pastoris strain for expressing CBCAS as well as construction method and application of recombinant pichia pastoris strain

Abstract

The invention discloses a recombinant pichia pastoris strain for expressing CBCAS as well as a construction method and application of the recombinant pichia pastoris strain, and relates to the technical field of biology. The construction method of the recombinant pichia pastoris strain comprises the following steps: connecting a coding gene as shown in SEQ ID NO.3 into an expression vector to obtain a recombinant plasmid; and introducing the recombinant plasmid into pichia pastoris competent cells, and screening to obtain positive recombinants, namely the recombinant pichia pastoris strain. The genome of the cannabinoid phenol content cannabinoid leaves is extracted, the CBCAS gene is obtained through PCR amplification, the CBCAS gene is subjected to heterologous expression in pichia pastoris, the cannabinoid phenolic acid synthetase with high catalytic activity is obtained, the conversion rate of the substrate cannabinoid phenolic acid to the cannabinoid phenol can be increased through the cannabinoid phenolic acid synthetase, and the CBCAS gene can be used for preparing the cannabinoid phenol. The waste caused by the fact that residual cannabinoid phenolic acid in the plants is not converted when various cannabinoids are extracted from the cannabis plants is avoided.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a recombinant Pichia pastoris strain expressing CBCAS, a construction method thereof, and an application thereof. Background Art

[0002] Cannabinoids are a class of secondary metabolites unique to cannabis. Among the currently known more than 140 cannabinoids, cannabichromene (CBC) has attracted attention because it does not have psychoactive properties and has good medicinal value. Recent studies have shown that CBC has good anticonvulsant effects and exhibits cytotoxicity to some cancer cells. The synergistic effect of CBC with other cannabinoids shows better therapeutic effects. However, due to the low expression level and catalytic activity of cannabichromenic acid synthase (CBCAS), the key enzyme for synthesizing CBC in cannabis plants, the content of CBC in cannabis plants is low, and it is costly and difficult to directly extract CBC from cannabis plants.

[0003] The yeast expression system was discovered in the 1970s of the last century and has been emphasized because of its advantages such as simple induction operation, short production cycle, low cost of culture medium, high protein expression level, etc. Pichia pastoris is a methylotrophic yeast that can use methanol as the sole carbon source, and has advantages such as protein secretion expression and post-translational modification of proteins, and is highly favored in the expression of foreign proteins.

[0004] In cannabis plants, CBC is generated from the substrate cannabigerolic acid (CBGA) under the catalytic action of the CBCAS enzyme to form cannabichromenic acid (CBCA), and CBCA is then converted by decarboxylation under natural conditions. Finding a method to increase the yield of its key enzyme, CBCAS, may be an effective way to solve the problem of low CBC yield. Therefore, it is necessary to develop a Pichia pastoris strain with high-yield cannabichromenic acid synthase to lay a foundation for the industrial production of CBCAS. Summary of the Invention

[0005] The object of the present invention is to provide a recombinant Pichia pastoris strain expressing CBCAS, a construction method thereof, and an application thereof to solve the problems existing in the above-mentioned prior art. Using this recombinant Pichia pastoris strain, a cannabichromenic acid synthase with high catalytic activity can be prepared, and this cannabichromenic acid synthase can increase the conversion rate of the substrate cannabigerolic acid to cannabichromene.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a cannabichromenic acid synthase, and the amino acid sequence of the cannabichromenic acid synthase is as shown in SEQ ID NO.2.

[0008] The present invention also provides a coding gene for the above-mentioned cannabicyclolic acid synthase.

[0009] Furthermore, the nucleotide sequence of the coding gene is as shown in SEQ ID NO.1 or SEQ ID NO.3.

[0010] The present invention also provides a method for constructing a recombinant Pichia pastoris strain expressing cannabicyclolic acid synthase, comprising the following steps:

[0011] Connect the coding gene as shown in SEQ ID NO.3 into an expression vector to obtain a recombinant plasmid;

[0012] Introduce the recombinant plasmid into Pichia pastoris competent cells, and screen to obtain positive recombinants, which are the recombinant Pichia pastoris strains.

[0013] Furthermore, the expression vector is the pPIC9K vector.

[0014] Furthermore, the Pichia pastoris competent cells are Pichia pastoris GS115.

[0015] The present invention also provides a recombinant Pichia pastoris strain constructed according to the above construction method.

[0016] The present invention also provides the application of the above-mentioned recombinant Pichia pastoris strain in the preparation of cannabicyclolic acid synthase.

[0017] The present invention also provides the application of the above-mentioned cannabicyclolic acid synthase in improving the conversion efficiency of cannabigerolic acid to cannabicyclolic acid.

[0018] The present invention also provides a method for increasing the yield of cannabicyclol extracted from cannabis plants, comprising the steps of using the above-mentioned cannabicyclolic acid synthase to catalyze cannabigerolic acid in cannabis plants to generate cannabicyclolic acid, and then generating cannabicyclol through a decarboxylation reaction.

[0019] The present invention discloses the following technical effects:

[0020] The present invention extracted the genome of cannabis leaves with a high cannabicyclol content, obtained the CBCAS gene by PCR amplification, heterologously expressed the gene in Pichia pastoris, and obtained a cannabicyclolic acid synthase with high catalytic activity. This cannabicyclolic acid synthase can increase the conversion rate of the substrate cannabigerolic acid to cannabicyclolic acid, and avoid the waste caused by the residual cannabigerolic acid in the plant not being converted when extracting various cannabinoids from cannabis plants. Description of the Drawings

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required for use in the embodiments. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can be obtained based on these drawings.

[0022] Figure 1 The amplification result of the CBCAS gene; where M: DS2000 Marker; 1: Negative control without added template DNA; 2: Target gene CBCAS.

[0023] Figure 2 The vector map of pPIC9K-CBCAS.

[0024] Figure 3 The verification result diagram of pPIC9K-CBCAS colony PCR; where M: DS2000 Marker; 1: Positive control with pPIC9K-CBCAS plasmid as template DNA; 2: Negative control without added template DNA; 3: Single colony of pPIC9K-CBCAS on the 6 mg / mL G418 plate.

[0025] Figure 4 The result diagram of SDS-PAGE verifying the induced expression of CBCAS protein; where M: Non-prestained protein Marker; 1: Supernatant of lysed yeast cells transfected with pPIC9K vector; 2: Induced supernatant of yeast transfected with pPIC9K vector; 3: Supernatant of lysed yeast cells transfected with pPIC9K-CBCAS vector; 4: Induced supernatant of yeast transfected with pPIC9K-CBCAS vector.

[0026] Figure 5 The result diagram of Western blot detecting the expression of CBCAS enzyme; where M: Color prestained protein Marker; 1: Supernatant of lysed yeast cells transfected with pPIC9K vector; 2: Induced supernatant of yeast transfected with pPIC9K vector; 3: Supernatant of lysed yeast cells transfected with pPIC9K-CBCAS vector; 4: Induced supernatant of yeast transfected with pPIC9K-CBCAS vector.

[0027] Figure 6 The result diagram of CBCAS temperature gradient enzyme activity detection; where the empty vector control group refers to the reaction solution using the yeast transfected with pPIC9K empty vector; the different temperature treatment groups refer to the reaction solutions of CBCAS expressed by pPIC9K-CBCAS strains reacting at different temperatures.

[0028] Figure 7It is a graph showing the detection results of the enzyme activity of CBCAS with time gradient; among them, the empty vector control group uses the reaction solution transfected with the pPIC9K empty vector; the different time treatment groups refer to the reaction of the CBCAS reaction solution expressed by the pPIC9K-CBCAS strain at different times. Detailed implementation manners

[0029] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0030] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0031] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0032] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are only exemplary.

[0033] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, that is, they are intended to include but not limited to.

[0034] Example 1

[0035] 1. Materials and methods

[0036] 1.1 Materials and instruments

[0037] The culture media related to the present invention are as follows:

[0038] LB culture medium: 10 g / L of tryptone, 5 g / L of yeast extract powder, 5 g / L of sodium chloride, and 12 g / L of agar.

[0039] MD medium: 20 g / L agar, 20 g / L glucose, 13.4 g / L non - amino nitrogen source, and 4×10 -4 mL / L biotin.

[0040] BMGY medium: 20 g / L tryptone, 10 g / L yeast extract powder, 3.94 g / L dipotassium hydrogen phosphate, 12 g / L dipotassium hydrogen phosphate, 20 mL / L glycerol, 13.4 g / L non - amino nitrogen source, and 4×10 -4 mL / L biotin.

[0041] BMMY medium: 20 g / L tryptone, 10 g / L yeast extract powder, 3.94 g / L dipotassium hydrogen phosphate, 12 g / L dipotassium hydrogen phosphate, 13.4 g / L non - amino nitrogen source, and 4×10 -4 mL / L biotin.

[0042] Note: Kana or Amp antibiotics are prepared into a 50 mg / mL aqueous solution, filtered and sterilized, and then added to the sterilized LB medium at a volume ratio of 1:1000.

[0043] The pPIC9K vector and pGG C000 vector used in the present invention were purchased from Wuhan Miaoling Biotechnology Co., Ltd.; the Pichia pastoris competent cells were Pichia pastoris GS115, purchased from Shanghai Angyu Biotechnology Co., Ltd.; the cannabis materials were provided by the Economic Crop Research Institute of Heilongjiang Academy of Agricultural Sciences; the synthesis and sequencing of all primers were completed by Beijing Tsingke Biotechnology Co., Ltd.; the codon - optimized CBCAS gene was synthesized by Jiangsu Saisuofei Biotechnology Co., Ltd.; the content of CBC in the present invention was detected using an Agilent high - performance liquid chromatography analyzer, and the liquid chromatography conditions were as follows: chromatographic column: Shimadzu sil - 16C18 column (150 mm×4.6 mm×3 μm), column temperature: 30 °C; mobile phase: A is an aqueous solution containing 0.1% formic acid, B is an acetonitrile solution containing 0.1% formic acid; isocratic elution: 25% A, 75% B, retention time is 20 min; ultraviolet detector: 230 nm; flow rate: 0.7 mL / min; injection volume: 10 μL.

[0044] 1.2 Experimental methods

[0045] 1.2.1 Cannabis genome extraction

[0046] Weigh the cannabis leaves with high CBC content, grind them in liquid nitrogen, and extract the total cannabis genome according to the instructions of the plant DNA extraction kit. Use NanoDrop2000 to detect the DNA concentration and quality. The cannabis genomic DNA is stored at - 20 °C for later use.

[0047] 1.2.2 Cloning of the CBCAS gene

[0048] Specific primers Primer 1 and Primer 2 were designed using the Primer 3plus online website (see Table 1). In this invention, a high-fidelity polymerase was selected for the amplification of the target fragment. Using cannabis genomic DNA as a template, add 10 μL of 5×SF Buffer, 1 μL of dNTPs, 2 μL each of Primer 1 and Primer 2, 2 μL of cannabis genomic DNA, and make up to 50 μL with deionized water in a PCR tube, and add 1 μL of Phanta Super-Fidelity DNA polymerase for PCR amplification. PCR amplification program: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 10 s, annealing at 56°C for 30 s, extension at 72°C for 2 min, for a total of 32 cycles; extension at 72°C for 5 min, and store at 4°C. After the PCR product was electrophoresed on a 1% agarose gel, the gel at the position of the target band was cut off and purified according to the instructions of the gel extraction kit.

[0049] Table 1 Primers used for vector construction and identification

[0050]

[0051]

[0052] Note: The underlines are the adapters of the primers.

[0053] Primers 3 and 4 containing the adapter of the pGG C000 vector were designed for high-fidelity amplification of the CBCAS gene. Add 1 μL of the empty vector pGG C000 to a PCR tube, add the purified product of the CBCAS gene so that the mass ratio of the CBCAS gene to pGG C000 reaches 3:1, 2 μL of 10×Cutsmart Buffer, 2 μL of BsaI, and make up to 20 μL with deionized water for PCR reaction. Specific reaction conditions: 37°C for 1 h; 80°C for 10 min; 4°C for 5 min; 25°C for 1 h; 70°C for 10 min; store at 4°C (add 2.5 μL each of T4 DNA ligase and T4 ligase Buffer during the 4°C, 5 min period). After the PCR reaction, take 10 μL of the ligation solution and add it to 50 μL of Escherichia coli DH5α competent cells, immediately ice-bath for 20 min; 42°C for 90 s; after ice-bathing for 3 min, shake at 37°C at 180 rpm for 60 min. Centrifuge at room temperature for 2 min, leave about 200 μL of the supernatant to resuspend the bacteria, evenly coat the bacterial solution on an LB plate containing Amp, and culture at 37°C for 12 h.

[0054] Pick monoclonal colonies on the plate for colony PCR verification. Add 7.5 μL of 2×Taq Max, 2 μL each of primer 3 and primer 4 into the PCR tubes, and make up to 20 μL with deionized water. Use a white pipette tip to pick different monoclonal colonies as templates and put them into each PCR tube. The specific reaction conditions are: 95°C for 3 min; 95°C for 20 s, 52°C for 30 s, 72°C for 2 min, for a total of 32 cycles; 72°C for 5 min; store at 4°C. The PCR products are detected by 1% agarose gel electrophoresis, with a sample loading volume of 10 μL, a constant voltage of 110 V, and electrophoresis for 25 min. Use a gel imager to record the results. For monoclonal colonies with the target band size, perform shaking culture, take 1 mL of the bacterial solution for sequencing. Obtain the sequence of the CBCAS gene according to the sequencing results. Optimize the codons of the CBCAS gene according to the usage preference of Pichia pastoris.

[0055] 1.2.3 Construction of yeast expression vector

[0056] Design primers 5 and 6 containing EcoR I and Not I restriction site linkers for high-fidelity amplification of the CBCAS gene. After the amplification products are electrophoresed on 1% agarose gel, cut the gel at the position of the target band and purify it according to the instructions of the gel extraction kit. Perform double digestion on CBCAS and pPIC9K vectors respectively. The digestion system is: 2 μL of 10×cutsmart buffer, 1 μL of EcoR I, 1 μL of Not I, 1.5 μg of the purified product of the CBCAS gene / pPIC9K vector, and make up to 20 μL with deionized water. Digest at 37°C for 4 h. After the digestion products are electrophoresed on 1% agarose gel, cut the gel at the position of the target band and purify it according to the instructions of the gel extraction kit. Use T4-DNA ligase to ligate the digested and purified CBCAS gene and pPIC9K vector. The ligation system is: 300 ng of the digested product of CBCAS, 100 ng of the digested product of pPIC9K vector, 1 μL of T4DNA ligase, 2 μL of 10×T4-DNA Buffer, and make up to 20 μL with deionized water. React at 16°C for 12 h. After the ligation products are purified by the product purification kit, transfer them into DH5α Escherichia coli competent cells, spread them evenly on the LB plate containing Amp resistance, and culture them inverted at 37°C for 12 h. Pick monoclonal colonies for colony PCR, and sequence the positive monoclonal colonies to confirm the successful construction of the pPIC9K-CBCAS vector.

[0057] 1.2.4 Yeast transformation and screening of positive recombinant bacteria

[0058] The codon-optimized recombinant plasmid pPIC9K-CBCAS was linearized with Sac I restriction endonuclease and then introduced into competent Pichia pastoris GS115 by electroporation. The cell suspension was evenly spread on MD solid medium and incubated at 30 °C in an inverted position for 3 - 5 days. The colonies on the plate were rinsed with deionized water and collected. 200 μL of the bacterial solution was pipetted and spread on YPD plates with different concentrations of geneticin (G418) (2, 4, 6 mg / mL) and incubated at 30 °C in an inverted position for 5 days. Yeast single colonies on the 6 mg / mL G418 plate were picked, and yeast genomic DNA was extracted using the high-temperature-ethyl acetate method; Primer 7 was designed in the CBCAS sequence, and Primer 8 was designed in the AOX1 terminator region of the pPIC9K vector. PCR identification was performed on the single colonies of the pPIC9K-CBCAS recombinant bacteria on the 6 mg / mL G418 plate using Primers 7 and 8. PCR reaction program: pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 20 s, annealing at 54 °C for 30 s, extension at 72 °C for 2 min, for 32 cycles; extension at 72 °C for 5 min, and stored at 4 °C. The PCR products were identified by 1% agarose gel electrophoresis and verified by sequencing. The positive pPIC9K-CBCAS strains were activated and stored at -150 °C in a refrigerator.

[0059] 1.2.5 Induced expression of recombinant protein

[0060] Picked positive pPIC9K-CBCAS recombinant bacteria were inoculated into YPD liquid medium respectively and cultured overnight at 30 °C and 230 r / min for activation; The activated bacterial solution was inoculated into 20 mL of BMGY medium at a volume ratio of 1% and cultured at 30 °C and 230 r / min until the OD 600 reached 2 - 6, the bacterial cell precipitate was collected by centrifugation at 4 °C for 10 min, the bacterial cells were resuspended with sterile water, the supernatant was removed by centrifugation at 4 °C for 5 min, and the cells were resuspended with BMMY medium until the OD 600 reached 1, then the yeast was induced to express at 30 °C and 230 r / min, and 1% volume ratio of methanol was supplemented every 24 h for continuous induction for 72 h, with 3 replicates in each group. The supernatant of the induced expression was taken for standby. The bacterial cell precipitate after induced expression was added with 20 mL of lysis buffer and sonicated for 30 min, centrifuged at 4 °C for 10 min, 20 mL of the supernatant was added to a 30 kD protein ultrafiltration tube, and centrifuged at 4 °C and 5000 rpm until about 500 μL of liquid remained in the upper tube. The crude extract in the upper tube was collected to obtain a concentrated crude protein extract, and the concentration was detected using a BCA protein concentration kit for standby.

[0061] 1.2.6 Western Blot analysis

[0062] The recombinant pPIC9K-CBCAS bacteria were separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane using the "sandwich method". The PVDF membrane was blocked with 5% skim milk for 2 h and the surface of the PVDF membrane was rinsed with TBST. The PVDF membrane was transferred to TBST containing His antibody (1:5000) and incubated overnight at 4°C. After shaking and washing 6 times with TBST (5 min each time), it was transferred to TBST containing HRP-labeled secondary antibody (1:4000) and incubated at room temperature for 2 h, followed by shaking and washing 8 times with TBST (5 min each time). Finally, the bands were detected using an ECL hypersensitive chemiluminescence detection kit and an imaging analyzer.

[0063] 1.2.7 Determination of recombinant protease activity

[0064] Take 0.1 g of hemp leaves with high CBGA content, add 50 mL of methanol and ultrasonically disrupt for 30 min. After detection by high performance liquid chromatography, a crude extract of CBGA with a concentration of 30.94 μg / mL was obtained and stored at -20°C for later use.

[0065] Take 50 μg of the concentrated crude protein extract (preparation method is shown in 1.2.5), use 50 μL of the crude extract of hemp leaves with high CBGA as the substrate, and supplement it to 500 μL with 0.1 M citric acid-sodium citrate buffer (pH 5.0). Incubate statically at 25°C, 30°C, 35°C, and 40°C for 16 h respectively to determine the optimal reaction temperature; incubate statically at 0 h, 2 h, 4 h, 8 h, 12 h, and 16 h respectively at the optimal reaction temperature to determine the optimal reaction time. After the reaction, add 600 μL of methanol to terminate the reaction, invert and mix well, centrifuge at 10000 rcf for 3 min, take the supernatant and filter it through a 0.22 μm filter membrane, and use a high performance liquid chromatograph to detect the production amount of CBC. Each group was repeated three times.

[0066] 2 Results and discussion

[0067] 2.1 Extraction of hemp genomic DNA and amplification of CBCAS gene

[0068] Using hemp genomic DNA as a template, the target gene CBCAS was amplified by PCR using primer 1 and primer 2. After 1% agarose gel electrophoresis of the PCR product, the bands were as Figure 1 shown. It can be seen that there is a bright single band in the figure. After ligating it to the intermediate vector pGG C000 and sequencing, the full length of the CBCAS gene was determined to be 1635 bp, encoding a total of 545 amino acids, which is the same length as the CBCAS gene (GenBank accession number: MW561076.1) submitted in the NCBI database, with 18 nucleotide differences, resulting in 11 amino acid differences. It can be seen that the present invention has discovered a new CBCAS protein.

[0069] The sequence of the CBCAS gene is shown in SEQ ID NO.1, and the amino acid sequence of the CBCAS protein is shown in SEQ ID NO.2.

[0070] SEQ ID NO.1:

[0071]

[0072] SEQ ID NO.2:

[0073] MNCSTFSFWFVCKIIFFFLSFNIQISIANPQENFLKCFSEYIPNNPANPKFIYTQHDQLYMSVLNSTIQNLRFISDTTPKPLVIVTPSNNSHIQATILCSKKVGLQIRTRSGGHDAEGMSYISQVPFVVVDLRNMHSIKIDVHSQTAWVEAGATLGEVYYWINEMNENFSFPGGYCPTVGVGGHFSGGGYGALMRNYGLAADNIIDAHLVNVDGKVLDRKSMGEDLFWAIRGGGGENFGIIAAWKIKLVVVPSKATIFSVKKNMEIHGLVKLFNKWQNIAYKYDKDLMLTTHFRTRNITDNHGKNKTTVHGYFSSIFLGGVDSLVDLMNKSFPELGIKKTDCKELSWIDTTIFYSGVVNYNTANFKKEILLDRSAGKKTAFSIKLDYVKKLIPETAMVKILEKLYEEEVGVGMYVLYPYGGIMDEISESAIPFPHRAGIMYELWYTATWEKQEDNEKHINWVRSVYNFTTPYVSQNPRLAYLNYRDLDLGKTNPESPNNYTQARIWGEKYFGKNFNRLVKVKTKADPNNFFRNEQSIPPLPPRHH。

[0074] 2.2 Construction of Yeast Expression Vector

[0075] The nucleotide sequence of the optimized CBCAS gene according to the codon preference of Pichia pastoris is shown in SEQ ID NO.3. Using this optimized sequence as a template, the CBCAS gene was amplified with high fidelity using primers 3 and 4 that add EcoR I and Not I restriction sites. After digestion, ligation, and transformation of the PCR product and the empty vector, electrophoresis was performed using primers 5 and 4. After determining that the position of the target band was correct, the plasmid was extracted for sequencing identification to confirm the successful construction of the pPIC9K-CBCAS vector (see the vector map in Figure 2 ).

[0076] SEQ ID NO.3:

[0077]

[0078] 2.3 Pichia pastoris transformation and screening of positive recombinant bacteria

[0079] The plasmid pPIC9K-CBCAS was linearized with Sac I and electrotransformed into competent Pichia pastoris GS115 cells. The cells were cultured at 30 °C for 4 days, and then high-copy positive bacteria were screened using different concentrations of G418. Single colonies on 6 mg / mL G418 plates were picked, and the yeast cells were treated by the high-temperature-ethyl acetate method. PCR amplification was performed using primer 7 designed based on the CBCAS gene sequence and primer 8 designed based on the terminator fragment of the pPIC9K vector. A 1380 bp band ( Figure 3 ) was obtained, indicating that the plasmid pPIC9K-CBCAS had been integrated into the chromosome of Pichia pastoris GS115.

[0080] 2.4 Induced expression of recombinant protein

[0081] The induced expression product of Pichia pastoris transformed with the pPIC9K vector was used as a negative control for SDS-PAGE analysis. As Figure 4 shown, a specific band was found in the supernatant of the sonicated cells, but no obvious protein band was found in the induced supernatant. It was speculated that most of the CBCAS protein was not secreted extracellularly.

[0082] 2.5 Western blot verification of recombinant protein expression

[0083] Western Blot analysis showed that the recombinant protein CBCAS was normally expressed in Pichia pastoris, with a specific band at 72 kD, while there was no expression in the yeast cells transformed with the empty vector ( Figure 5 ). The molecular weight was slightly larger than the predicted protein molecular weight (62.1 kD) using the Protparam online software and the molecular weight of the CBCAS enzyme treated with endoglycosidase by Kaitlin et al. (63 kD). It was speculated that the CBCAS protein was glycosylated during yeast expression, resulting in an increase in molecular weight.

[0084] 2.6 HPLC detection of CBCAS enzyme activity

[0085] In this invention, the crude yeast protein extract from Pichia pastoris transformed with the pPIC9K vector was used as a negative control. The enzyme activity was determined by using the crude CBGA extract (CBGA content: 30.94 μg / mL) extracted from cannabis leaves with high CBGA content as a substrate and detecting the production amount of CBC using a high-performance liquid chromatograph.

[0086] Since the crude CBGA extract contains a small amount of CBC, CBC can also be detected when no enzyme solution is added and when the crude extract of empty bacterial protein is added. Therefore, the following method is used to calculate the amount of CBC generated: CBC generation = CBC content in the treatment group - CBC content in the group without enzyme solution added.

[0087] In order to determine the optimal reaction conditions of CBCAS, the enzyme activity was measured at a temperature gradient of 25℃, 30℃, 35℃, and 40℃ for 16h to determine the optimal reaction temperature of CBCAS enzyme in CBGA crude extract. The results showed that the amount of CBC generated in the reaction system reached a maximum of 52.3ng / mL at 30℃; when reacting at 35℃ and 40℃, the CBC content of the three treatment groups was lower than that of the three treatment groups at 25℃ and 30℃ ( Figure 6 ). The above results show that the optimal activity temperature of the CBCAS enzyme of the present invention is 30°C.

[0088] Then, the enzyme activity was measured at 30°C for 4h, 8h, 12h, and 16h to determine the reaction time when the enzyme activity reached the maximum. It was found that when the reaction was carried out for 12h, the CBC content in the system reached 256.8ng / mL, reaching the highest value among several groups of reactions, significantly higher than the control group without enzyme solution (179.1ng / mL) and the control group with pPIC9K empty carrier protein added (180.5ng / mL), and the newly generated CBC reached 77.7ng / mL.

[0089] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A cannabidiol acid synthase, characterized in that The amino acid sequence of the cannabidiol acid synthase is shown in SEQ ID NO.

2.

2. A gene encoding the cannabidiol acid synthase as claimed in claim 1.

3. The coding gene according to claim 2, characterized in that The nucleotide sequence of the coding gene is shown as SEQ ID NO.1 or SEQ ID NO.

3.

4. A method for constructing a recombinant Pichia pastoris strain expressing cannabidiol acid synthase, characterized in that: The following steps are involved: Connect the coding gene shown in SEQ ID NO.3 into the expression vector to obtain a recombinant plasmid; The recombinant plasmid is introduced into Pichia pastoris competent cells, and positive recombinants are obtained by screening, namely the recombinant Pichia pastoris strain.

5. The construction method according to claim 4, characterized in that: The expression vector is a pPIC9K vector.

6. The construction method according to claim 4, characterized in that: The Pichia pastoris competent cell is Pichia pastoris GS115.

7. A recombinant Pichia pastoris strain constructed according to the construction method according to any one of claims 4 to 6.

8. Use of the recombinant Pichia pastoris strain as claimed in claim 7 in the preparation of the cannabidiol acid synthase as claimed in claim 1.

9. Use of the cannabichromenic acid synthase as claimed in claim 1 in improving the conversion efficiency of cannabichromenic acid to cannabichromenic acid.

10. A method for increasing the yield of cannabidiol extracted from cannabis plants, characterized in that: The method comprises the steps of using the cannabicyclopentyl acid synthase described in claim 1 to catalyze the cannabicyclopentyl acid in the cannabis plant to generate cannabicyclopentyl acid, and then generating cannabicyclopentyl phenol through a decarboxylation reaction.

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